E2 is the major viral regulatory protein of the papillomaviruses (PV). To understand the basis for persistence of these pathogenic viruses in humans and their association with malignancies, a detailed analysis of E2 function is needed. This proposal investigates the protein:protein interactions which are critical for E2 function. The long term objective is a comprehensive understanding of the processes which mediate the transcriptional activation and replication functions of E2. This will include identifying the cellular proteins which interact with E2. The goal of the proposed research is to isolate human cDNAs which encode proteins that interact with the human papillomavirus type 18 (HPV-18) E2. This will be achieved through the use of a yeast "two hybrid" screen which has been successfully used to isolate a novel cellular protein which interacts with bovine papillomavirus (BPV) E2. The physical association between 'candidate' cDNA encoded proteins and E2 will be investigated in vitro. This will be done by co-immunoprecipitations with E2 specific antibodies and the use of recombinant GST fusion peptides. The functional significance of interactions will also be tested by creating mutant E2 proteins that have lost the ability to interact with the factor in question. This will be done by random mutagenesis of particular regions of the E2 coding sequence to create mutant libraries, followed by phenotypic screening in yeast for non- interacting mutants. Any such mutants will be tested for transcriptional activation and transient replication functions by transfection into human cells.